The goal of this R01 project is to identify allosteric inhibitors that trap inactive conformations of a family of proteolytic enzymes and have sufficient pharmacologic viability to serve as the starting point for lead optimization and animal studies. These studies seek to exploit dynamics associated with monomer-dimer equilibrium to offer a new approach for developing specificity against this class of protease targets that has so far been recalcitrant to selective inhibitors. Protein-protein interactions are ubiquitous in biology ad represent potential therapeutic targets for numerous diseases. The dimeric human herpes virus (HHV) proteases are one such example. HHVs make up one of the most prevalent viral families and are the etiological agents to a variety of devastating human illnesses for which there is lack of specific and effective treatments. As with all infectious diseases, resistance to therapy is constantly evolving and new therapies are needed for this virus family. There is significant interest in new viral protease inhibitors based on the recent success of antiproteolytic therapies. All HHVs express a dimeric serine protease that is essential to the viral life cycle. Genetic knockout of this protease in cell culture prevents viral replication, providing genetic validation f the target. We have identified a small molecule inhibitor of the protease of one member of this family, Kaposi's sarcoma-associated herpes virus (KSHV), by screening a biased helical mimetic library. By integrating multiple chemical-biology approaches we have determined a dimer disruption via monomer trap mode of inhibition and mapped the binding site to a previously unreported allosteric pocket at the protease dimer interface. Recent chemistry efforts have improved potency and permeability while further informing mode of binding. Considering the structural and functional homology among HHV proteases, we propose to use diverse screening approaches and structure-based inhibitor design to develop allosteric inhibitor scaffolds that target protease dimerization or other allosteric sites in herpes virus proteases. These assays, including cell culture assays for viral infectivity, are currently in place for KSHV protease and CMV protease and will be established for other HHV proteases. We hypothesize that allosteric inhibitors of HHV proteases that trap inactive protease conformations can be identified and used to develop pharmacologically-viable compounds that prevent viral replication in cell-based assays. Aim 1. Develop inhibitory scaffolds for HHV proteases using screening and structure-guided chemistry to achieve nanomolar inhibition and improved cell membrane permeability. Aim 2. Characterize the specificity and binding mode of screening hits using NMR spectroscopy and crystallography and select allosteric inhibitors with a broad spectrum of activity towards KSHV, CMV, and HSV-2 proteases. Aim 3. Determine the mechanism of action of selected inhibitors in herpes viral cell culture models.